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We replaced the URA2 gene by six different deleted alleles constructed in vitro by Bg/II digestion in order to correlate the genetic map with the restriction map and to define the regions coding for the different functions of the carbamylphosphate synthetase--aspartate transcarbamylase complex (CPSase-ATCase). We also enlarged the collection of ura2 point mutations by using a positive selection method based on resistance to the toxic accumulation of ureidosuccinic acid (USA). Of the new independent mutations nine mapped in the intermediary zone, a previously defined mutationless region localized between regions coding for CPSase and ATCase. This shows that the former definition resulted from analysis of a limited number of mutants (40). The study of an allele deleted in the intermediary zone shows that this sequence codes for a protein region necessary for the feedback inhibition of the CPSase-ATcase enzyme complex. The CPSase- ATCase- phenotype of 26 mutants resistant to USA accumulation shows the importance of the in vivo channelling of carbamylphosphate in the CPSase-ATCase complex for USA and subsequent pyrimidine biosynthesis. Finally, our results confirm that the CPSase and ATCase activities are separate functions.

Transcription of the two unlinked structural genes URA1 and URA3 of Saccharomyces cerevisiae is positively regulated by the gene product PPR1. We have used S1 digestion and primer extension mapping to investigate the RNAs produced in different genetic backgrounds: wild-type, ppr1 deletion mutants, constitutively induced and non-inducible ppr1 mutants. Results show that each structural gene specifies multiple messenger RNA classes with different 5'-terminal sequences. The basal level of these transcripts does not require a functional PPR1 gene. Induction of URA1 results from an even increase of the level of synthesis of all the transcripts in contrast to that of URA3 which is effected by selectively increasing the levels of synthesis of one subset of transcripts. The PPR1-mediated control was also studied in the foreign genetic background of Schizosaccharomyces pombe using autonomously replicating hybrid plasmids carrying the gene URA1 or URA3 along with the regulatory gene PPR1, either in a constitutive or non-inducible allelic form. The 5' ends of the transcripts URA1 and URA3 made in S. pombe map upstream from the initiation sites used in S. cerevisiae. In contrast to S. cerevisiae, in S. pombe the URA3 but not URA1 transcripts respond to the PPR1-induction. We have identified a minimal control region for the PPR1-specific induction of URA1, that includes sequences located between the T-A-T-A box and the translation start codon. This region contains sequence features in common with URA3. There is an extensive alternating Pu:Py region including the T-A-T-A box of both promoters and an eight base-pair exact homology; further downstream, there is another 11 base-pair highly conserved sequence which either overlaps or lies in close proximity to the unregulated start sites of URA1 in S. pombe and of URA3 in S. cerevisiae. A positive regulatory model taking into accounts all these observations is presented.

A pool of random wild type yeast DNA fragments obtained by partial Sau IIIA restriction enzyme digestion and inserted in the Bam HI site of the hybrid yeast Escherichia coli plasmid ((pFL1) has been used to transform to prototrophy a methionyl-tRNA synthetase-impaired mutant requiring methionine. In the numerous prototroph strains recovered at least two independent clones have been obtained which show nonchromosomic inheritance character and an approximately 30-fold increase in methionyl-tRNA synthetase activity as compared to the wild type. Measurement of the Km for methionine in the transformed yeast cells indicates that the activity has been restored by decreasing the Km for methionine to the same level as found for the wild type methionyl-tRNA synthetase. Southern blotting experiments show that the yeast DNA's fragments inserted in the two independent plasmids share a common sequence which must correspond at least partly to the structural gene for methionyl-tRNA synthetase. They also suggest that the methionyl-tRNA synthetase gene is differently orientated in the two plasmids

A cDNA sequence coding for chicken ovalbumin was fused to the beginning of the Escherichia coli lactose operon and recombined in vitro with a composite vector plasmid which can be propagated in both E. coli and Saccharomyces cerevisiae. Such plasmids direct the synthesis of an ovalbumin-like protein (OLP) in both microorganisms, probably because the E. coli lac regulatory region has some promoter activity in yeast. The yeast strains produce about 1 000 to 5 000 molecules of ovalbumin-like protein per cell.

S1 mapping on the VAS1 structural gene indicates the existence of two classes of transcripts initiating at distinct in-frame translation start codons. The longer class of VAS1 transcripts initiates upstream of both ATG codons located 138 base pairs away and the shorter class downstream of the first ATG. A mutation that destroys the first AUG on the long message results in respiratory deficiency but does not affect viability. Mutation of the ATG at position 139 leads to lethality because the initiating methionine codon of the essential cytoplasmic valyl-tRNA synthetase has been destroyed. N-terminal protein sequence data further confirm translation initiation at ATG-139 for the cytoplasmic valyl-tRNA synthetase. From these results, we conclude that the VAS1 single gene encodes both mitochondrial and cytoplasmic valyl-tRNA synthetases. The presequence of the mitochondrial valyl-tRNA synthetase shows amino acid composition but not the amphiphilic character of imported mitochondrial proteins. From mutagenesis of the ATG-139 we conclude that the presequence specifically targets the cytoplasmically synthesized mitochondrial valyl-tRNA synthetase to the mitochondrial outer membrane and prevents binding of the enzyme core to cytoplasmic tRNAVal.

The URA4 gene of Saccharomyces cerevisiae, coding for the third enzyme of the pyrimidine pathway, has been cloned through phenotypic complementation of a ura4 mutant of S. cerevisiae. Subcloning of an original 9 kb DNA fragment, carrying the yeast URA4 gene, allowed us to localize the gene on a 2 kb ClaI--BamHI fragment. The sequence of the URA4 structural gene and surrounding DNA was determined by the dideoxynucleotide chain termination method. The URA4 gene encodes a dihydroorotase subunit of calculated molecular weight 40,600. S1 nuclease mapping indicated that transcription of URA4 is initiated at four major start sites located at positions -41, -30, -22 and -18. A set of potentially significant sequences was identified in the 5' OH non-coding region of the gene. The deduced amino acid sequence of dihydroorotase was examined and compared with homologous amino acid sequences of Salmonella typhimurium, Escherichia coli and Drosophila melanogaster. S. cerevisiae dihydroorotase shows 40% homology with the S. typhimurium and E. coli enzymes and 23% homology with the D. melanogaster enzyme. A potential active site has been predicted for dihydroorotase from these comparisons.

Yeast URA2 encodes a multifunctional carbamoyl phosphate synthetase-aspartate transcarbamylase of 220,000 molecular weight. We determined the nucleotide sequence of the 5' proximal part of the gene which is responsible for the glutamine amide transfer function of the carbamoyl phosphate synthetase activity. Alignment of the enzyme sequence derived from URA2 with sequences from Escherichia coli carA carB and yeast arginine-specific CP A1 CP A2 indicates that monofunctional and bifunctional carbamoyl phosphate synthetases are probably homologous. The URA2-derived enzyme organization is NH2-carbamoyl phosphate synthetase-aspartate transcarbamylase-CO2H.

We selected a 5-fluorouracil-resistant, thermosensitive mutant of the uridine monophosphokinase step in Saccharomyces cerevisiae. The mutant displays very weak thermolabile uridine monophosphokinase activity and wild-type uridine diphosphokinase activity. Growth of the mutant at the non-permissive temperature causes immediate reduction of pyrimidine triphosphate pools to 10% of the wild-type level as well as significantly lowering total RNA and protein synthesis. These conditions also provoke derepression of the first gene of the pathway, URA2, at both the levels of enzymatic activity and transcription. The mutation segregates independently of all known genes of the pyrimidine biosynthetic pathway. The corresponding gene has been isolated on a 4.8 kb fragment by complementation of the mutant phenotype. The new gene, named URA6, codes for a 2.2 kb polyadenylated messenger RNA, exists in a single copy per haploid genome, and was mapped to the centromere of chromosome XI.